E3 Ubiquitin Ligase NEDD4L Regulates TGF-β1/Smad Signaling Pathway to Mediate High-glucose and High-fat-induced Ferroptosis of Hepatocytes

Objective: To explore the molecular mechanism of E3 ubiquitin ligase neural precursor cell-expressed developmentally down-regulated 4-like (NEDD4L) regulating high-glucose and high-fat-induced ferroptosis in hepatocytes via modulation of transforming growth factor (TGF)-β1/Smad signaling pathway. Methods: Hepatocytes THLE-2 were cultured in high-glucose and high-fat medium to establish an in vitro non-alcoholic fatty liver disease (NAFLD) model. This study detected cellular lipid deposition, cell viability, cellular superoxide dismutase (SOD) activity, glutathione (GSH), malondialdehyde (MDA), ferrous iron (Fe2+), reactive oxygen species (ROS) levels, and cellular mitochondrial membrane potential (MMP). Meanwhile, cellular NEDD4L, GPX4, ACSL4, SLC7A11, TGF-β1, TβRII and p-Smad2/3 levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. In addition, TGF-β1-TβRII and NEDD4L-TβRII interactions were evaluated by Co-immunoprecipitation. Results: High-glucose and high-fat treatment led to ferroptosis in hepatocytes, manifested by decreased cell viability, SOD activity and GSH level, increased MDA, Fe2+ and ROS levels, and reduced MMP. High-glucose and high-fat treatment downregulated NEDD4L expression in hepatocytes; by contrast, overexpression of NEDD4L alleviated ferroptosis in hepatocytes. NEDD4L inhibited TGF-β1 signaling by mediating TβRII ubiquitination and degradation. Besides, suppressed TGF-β1/Smad signaling pathway alleviated ferroptosis in hepatocytes, and NEDD4L could regulate hepatocyte ferroptosis by mediating TGF-β1/Smad signaling pathway. Conclusion: NEDD4L can inhibit high-glucose and high-fat-induced ferroptosis in hepatocytes through suppressing the TGF-β1/Smad signaling pathway via mediating TβRII ubiquitination and degradation.